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Design and construction of a synthetic CO2 fixation cycle in vitro

  • Author(s): Luo, Shanshan
  • Advisor(s): Liao, James C
  • et al.
Abstract

The increasing atmospheric carbon dioxide (CO2) level is calling for more efficient CO2 fixation systems to re-balance the carbon cycle. At the same time, atmospheric CO2 can in principle be a cheap and abundant carbon source for synthesizing various organic compounds. Converting CO2 into useful chemicals can be an ideal solution for a sustainable future. However, due to its high thermodynamic stability and kinetic inertness, the chemical transformation of CO2 into organic compounds usually requires quite harsh conditions. In contrast, nature has evolved several CO2 fixation pathways for synthesizing longer carbon chain compounds from CO2 under ambient conditions. To date, six CO2 fixation pathways have been reported. However, they all contain key carboxylases with intrinsic properties such as structural complexity and oxygen sensitivity that hinder their further engineering and applications. To circumvent these problems, we designed a synthetic CO2 fixation cycle composed of all efficient, simple and oxygen tolerant enzymes and demonstrated its function in vitro.

In this work, we utilized the two most active and oxygen-insensitive carboxylases, crotonyl-CoA carboxylase/reductase (Ccr) and phosphoenolpyruvate carboxylase (Ppc) to fix CO2 and bicarbonate, respectively. Our designed cycle is composed of the reductive glyoxylate synthesis (rGS) pathway and the reductive pyruvate synthesis (rPS) pathway. The rGS pathway converts one pyruvate to one glyoxylate and one acetyl-CoA fixing one bicarbonate, while the rPS pathway converts one acetyl-CoA to pyruvate with one CO2 fixed. Together, we obtained a CO2 fixation cycle termed the reductive glyoxylate/pyruvate synthesis (rGPS) cycle.

We first validated the feasibility of rGPS with its stepwise reconstitution in vitro. Using a two-pot immobilized enzyme system, we were able to run the rGPS cycle semi-continuously. To further improve its efficiency, we developed electrochemical and enzymatic FAD regeneration systems to run the rGPS cycle continuously in one pot. Using the enzymatic FAD regeneration method increased the CO2 fixation rate of rGPS to 66.9 nmol min-1 mg-1 of core cycle proteins.

We further explored the construction of rGPS in vivo. The rGS part has been demonstrated in vivo. The rPS part has not worked in E. coli for growth rescue, although all ten enzymes of rPS were confirmed to be active in crude extract assays. Finally, strategies for constructing a fully functional rGPS in vivo have been proposed.

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